Hemicellulose and hemicellulases: Hemicelluloses have a complex chemical structure and are often referred to as mannans, xylans and galactans on the basis of the predominant sugar type in the main chain. A range of mannan-type polysaccharides is synthesised by a wide variety of plants and is found in different types of plant tissue.
The main role of hemicelluloses and galactomannans is to function as structural polysaccharide and/or as reserve energy. Besides amylose and amylopectin which are the most widespread storage polysaccharides in plants, there is a diverse group of mannan-based polysaccharides found in seeds, roots, bulbs and tubers of various plants. These include mannans, galactomannans and glucomannans. Mannans are comprised of linear chains of β-1-4-mannan and are found in the plant seed endosperm of certain plant species. Mannan has been isolated from ivory nut, date or green coffee bean. In most cases the mannans are highly insoluble in water and very dense. Accordingly, it has been suggested that the mannan forms the molecular basis for the hardness which is characteristic for palm kernels, such as ivory nut. Galactomannans are reserve polysaccharides in the seed endosperm of leguminous plants. In contrast to unsubstituted mannans, the galactomannans are water soluble and can store water in the seed. Polysaccharides can be organised in structures ranging from irregular amorphous to highly organised crystalline structures. Crystalline linear β-(1-4)-D-mannan has for example been found in the cell walls of ivory nuts in two morphologies, mannan-I and mannan-II, respectively. The former is of granular morphology, while the latter is fibrillar.
Due to the complex structural composition of the plant cell wall, microorganisms thriving on decaying plant material must possess a number of different enzymes that are able to hydrolyse these highly polymeric and mostly insoluble materials. These enzyme systems often comprise a combination of endo- and exo-acting enzymes. The two major endo-acting enzymes involved in degradation of hemicelluloses are β-mannanase and β-xylanase. In addition, the exo-acting enzymes β-mannosidase, α-galactosidase and β-glucosidase are needed for complete degradation of galactoglucomannan. Often these enzyme have a modular structure in which a catalytic domain is connected through a linker region to a carbohydrate-binding domain (CBD) (Warren (1996) Microbial hydrolysis of polysaccharides. Annu. Rev. Microbiol., 50:183-212). However, other domains such as thermostabilising modules have been found in some xylanases.
Mannanases: Endo-β-1,4-D-mannanase (β-mannanase; EC 3.2.1.78) catalyses the random hydrolysis of manno-glycosidic bonds in mannan-based polysaccharides. Most β-mannanases degrade oligosaccharides down to DP4 (Biely and Tenkanen (1998) Enzymology of hemicellulose degradation, pages 25-47. In Harman and Kubiceck (ed) Trichoderma and Gliocladium, vol. 2, Taylor and Francis Ltd. London), however, residual activity has been demonstrated on mannotriose, indicating at least four subsites for mannose binding on the protein. The main end products of hydrolysis are often mannobiose and mannotriose, although significant amounts of mannose are also produced. Some β-mannanases are able to degrade crystalline mannan. In addition to hydrolysis, several β-mannanases including β-mannanase from Trichoderma reesei, have been shown to form transglycosylation products with either mannose or mannobiose as glycosidic bond acceptor.
β-mannanases have been isolated from a wide range of organisms including bacteria, fungi, plants and animals. Although mostly extracellular, some β-mannanases appear to be cell-associated. Their expression is often induced by growth on mannan or galactomannan, however, β-mannanase from T. reesei can also be induced by cellulose, while its expression is suppressed by glucose and other monosaccharides. Frequently multiple mannanases with different isoelectric points are found in the same organism, representing products from different genes or different products from the same gene, respectively.
Trichoderma reesei produces a potent enzyme system for the degradation of hemicellulase (Biely and Tenkanen (1998) Enzymology of hemicellulose degradation, pages 25-47. In Harman and Kubiceck (ed) Trichoderma and Gliocladium, vol. 2, Taylor and Francis Ltd. London), including the β-mannanase Man5A, which is transcribed from the gene man1 (Stalbrand et al. (1995) Cloning and expression in Saccharomyces cerevisiae of a Trichoderma reesei β-mannanase gene containing a cellulose binding domain. Appl. Environ. Microbiol. 61:1090-1097). Isoforms with varying isoelectric points are thought to represent different posttranslational modifications such as glycosylation. The mannanase consists of a N-terminal catalytic domain and a C-terminal cellulose-binding domain which are connected via a highly O-glycosylated Thr/Pro-rich linker. The structure of the catalytic domain has been solved and reveals a globular domain, belonging to the (βα)8-barrel structural class (Sabini et al. (2000) The three-dimensional structure of Trichoderma reesei β-mannanase from glycoside hydrolase family 5. Acta Crystallogr. Sect. D: Biol. Crystallogr. 56:3-13). A shallow substrate binding groove could be identified, and two additional β-sheets which are not conserved in other (βα)8-barrel type proteins.
In general, β-mannanases have moderate temperature optima between 40° C. and 70° C., except some β-mannanases from thermophiles (Politz et al. (2000) A highly thermostable endo-1,4-β-mannanase from the marine bacterium Rhodothermus marinus; Appl. Microbiol. Biotechnol. 53:715-721). The pH-optimum is in the neutral or acidic region, e.g. pH 5.0 for β-mannanase from T. reesei (Arisan-Atac et al. (1993) Purification and characterisation of a β-mannanase of Trichoderma reesei C-30; Appl. Microbiol. Biotechnol. 39:58-62). The molecular weights of the enzymes range between 30 kD and 80 kD.
Enzyme stability: Stability (incl. thermostability, pH stability and stability against proteolytic digestion) and activity under application conditions is a critical parameter for many industrially applied enzymes, since these enzymes often tend to be insufficiently stable or active under application conditions. For example, high thermostability allows a lower dosage of the enzyme due to longer activity under high temperature process conditions and therefore provides a commercial advantage. Many mesophilic and thermophilic organisms express enzyme variants which are adapted to the respective temperature conditions in which the organisms thrive. While mesophilic species tend to have less thermostable enzymes, thermophilic organisms must possess highly stable enzymes. A comparison of homologous enzymes from meso- and thermophilic organisms reveals two basic mechanisms for the increased stability. One is “structure-based” where the hyperthermophilic enzymes are significantly more compact than their mesophilic counterparts. Stability is enhanced by the sheer number of interactions. In contrast, a so-called “sequence-based” mechanism utilises a small number of apparently strong interactions which are responsible for the high thermal stability (Berezovsky and Shakhnovich (2005) Physics and evolution of thermophilic adaptation. Proc. Natl. Acad. Sci. USA 102:12742-12747). β-Mannanases from mesophilic (Braithwaite et Al. (1995) A non-modular endo-β-1,4-mannanase from Pseudomonas fluorescens subspecies cellulosa. Biochem. J. 305:1005-1010; Tamaru et al. (1995) Purification and characterisation of an extracellular β-1,4-mannanase from a marine bacterium Vibrio sp. strain MA-138, Appl. Environ. Microbiol. 61:4454-4458) and thermophilic bacteria (Duffaud et al. (1997) Purification and characterization of extremely thermostable beta-mannanase, beta-mannosidase and alpha-galactosidase from the hyperthermophilic eubacterium Thermotoga neapolitana 5068, Appl. Environ. Microbiol. 64:4428-4432; Gibbs et al. (1996) Sequencing, cloning and expression of a beta-1,4-mannanase gene, manA, from the extremely thermophilic anaerobic bacterium Caldicellulosiruptor Rt8B.4 FEMS Microbiol. Lett. 141:37-43; Bicho et al. (1991) The characterization of a thermostable endo-β-mannanase cloned from Caldocellum saccharolyticum. Appl. Microbiol. Biotechnol. 36:337-343) have been described.
Enzymes used in large amounts in technical processes must be produced at relatively low costs to allow a commercially viable process. This entails the availability of an efficient production host which produces and ideally secrets the target enzyme at several grams per litre culture. Heterologous expression of thermostable mannanases has been described in E. coli (Parker et al. (2001) Galactomannanases Man2 and Man5 from Thermotoga Species: Growth, physiology on galactomannans, gene sequence analysis and biochemical properties of recombinant enzymes. Biotechnol. Bioeng. 75:322-333) and S. cerevisiae (Stalbrand et al. (1995) Cloning and expression in Saccharomyces cerevisiae of a Trichoderma reesei β-mannanase gene containing a cellulose binding domain. Appl. Environ. Microbiol. 61:1090-1097) with very low yield. However, T. reesei mannanase has been expressed in Pichia pastoris at 10 g/l yield (Hägglund (2002) Thesis, Lund University, Sweden). On the other hand, Trichoderma reesei is a well known host for the production of a variety of industrial enzymes and homologous overexpression of mannanase or thermostable mannanase variants may result in high yield of a naturally processed enzyme.
Applications of mannanase enzymes: The use of mannanase enzymes is widespread in food & feed applications, the detergent and the pulp & paper industry. The use of mannanase enzymes as feed additives has been shown to provide several beneficial effects. These benefits are seen in combination with feedstuff like guar, copra, alfalfa, palm kernel and soy which contain significant amounts of mannans.
For monogastric animals like poultry and swine mannans are largely indigestible feed components that act as antinutritional factors. Negative effects of mannans reported are reductions in animal growth, feed efficiency and nutritional value of the feed (Lee et al. (2005) Effects of guar meal by-product with and without beta-mannanase Hemicell on broiler performance. Poult. Sci. 84:1261-1267).
Conversely, mannanase when added to e.g. corn-soybean meal diets for laying hens increased egg production and egg weight (Jackson et al. (1999) Effects of beta-mannanase in corn-soybean meal diets on laying hen performance. Poult. Sci. 78:1737-1741).
Mannanase enzymes are also used in combination with other carbohydrases in animal diets which has been shown to improve growth performance and nutrient digestibility (Kim et al. (2003) Use of carbohydrases in corn-soybean meal-based nursery diets. J. Anim. Sci. 81:2496-2504).
Mannanase enzymes added to corn-soybean meal diets for pigs improved the feed efficiency in late-nursery pigs. Furthermore, daily gain and feed efficiency was improved in growing-finishing pigs. Additionally, the mannanase enzyme increased the lean gain of finishing pigs (Pettey et al. (2002) Effects of beta-mannanase addition to corn-soybean meal diets on growth performance, carcass traits, and nutrient digestibility of weanling and growing-finishing pigs. J. Anim. Sci. 80:1012-1019).
In the food industry mannanase enzymes are described for the use in the production of instant coffee where the enzyme reduces the viscosity of the coffee extracts due to hydrolysis of the coffee mannan. This leads to reduced energy cost in the drying process involved in instant coffee production.
Mannanases are used to produce specific mannooligomers that are of interest as functional food ingredients. In particular mannooligomers with a prebiotic functionality are of interest in this application. In such applications plant derived mannopolymers are subjected to hydrolysis by mannanases.
Furthermore, mannanase enzymes are used in detergent compositions because of their mannan hydrolytic activity. Here the mannanases facilitate the removal of food and cosmetic derived stains/soils that often comprise mannan containing additives like stabilizers, emulsifiers and thickeners. These additives make up an important part of consumer relevant stains/soils.
For such detergent applications mannanases are also used in combination with other enzymes that are found in detergent formulations like amylases, cellulases, lipases, pectinases, proteases and endoglucanases.
In a more specific cleaning application mannanases are applied to remove biofilms from surfaces or tubings that need to be free from microbials like pharmaceutical equipment. In this application mannanases are often used in combination with detergents and other enzymes like carbohydrases and proteases.
Another application for mannanase enzymes is the enzyme-aided bleaching of paper pulp. Here mannanases can complement the action of xylanases that are traditionally used in enzyme-aided bleaching of pulp. Due to its different activity the mannanase hydrolyses e.g. the glucomannan parts of the pulp fibres that are not hydrolyzed by xylanases.
The effect of enzymatic treatment on pulp bleachability depends on the one hand on the wood species and on the other hand on the type of cooking (e.g. kraft pulp (sulfate cooking) or sulfite method). In this respect mannanase treatment was found to be especially effective in improving the bleachability of pulps produced by modified or continuous pulping methods that generally exhibit a low lignin content (Suurnakki et al. (1997) Hemicellulase in the bleaching of chemical pulps. Adv. Biochem. Eng. Biotechnol. 57: 261-287).
Moreover, mannanase enzymes are applied in the process of oil and gas well stimulation by hydraulic fracturing. Here mannanase enzymes reduce the viscosity of a process relevant guar solution that is, mixed with sand, applied in the application and needs to be liquefied in a certain stage of the application. As the process of hydraulic fracturing typically involves high temperatures, thermostable mannanases are desired (McCutchen et al. (1996) Characterization of Extremely Thermostable Enzymatic Breakers (Alpha-1,6-Galactosidase and Beta-1,4-Mannanase) from the Hyperthermophilic Bacterium Thermotoga-Neapolitana-5068 for Hydrolysis of Guar Gum. Biotechnology, and Bioengineering 52: 332-339).
Another application for mannanases is the controlled release of drugs or other material from matrices that are composed of cross linked galactomannans. It was for example shown that a chemically cross linked galactomannan hydrogel can serve as a colon specific drug delivery system in a way that the release of bacteria that secrete mannanases.
Most of the technical processes detailed above are performed at elevated temperatures, thus enzymes with a high thermal stability are preferable. Although thermostable mannanases have been described, they potentially suffer from poor technical producibility. Trichoderma reesei is well established for the high yield production of proteins, in particular homologous proteins, yielding tens of grams per litre culture. Thermostability is also required for feed additives that are incorporated in the feed mixtures prior to a pelleting procedure that comprises high temperatures. Additionally, mannanases applicable as feed additives need to be low-pH- and pepsin-stable and have to be active at low pH in order to be able to work efficiently in the stomach of e.g. monogastric animals.
In USRE38,047 a hemicellulase supplement is disclosed which serves to improve the energy efficiency of hemicellulose-containing animal feed. However, despite being from bacterial origin (Bacillus), the said hemicellulase is a wild type hemicellulase lacking improved properties introduced into the enzyme by design. Above all, the said hemicellulase has only limited thermostability. When treated with a temperature above 60° C., enzyme activity drops significantly. This is a severe disadvantage when it comes to the pre-treatment of animal feed. Here feed pelleting is an important process which improves supply of feed as well as the quality in terms of digestibility and microbial load. Microbial load is significantly reduced at elevated pelleting temperature, e.g. 70° C. Due to the fact that the said mannanase features only limited heat stability, the mannanase has to be added to the feed after pelleting. This is usually accomplished by spraying the said mannanase on the pellets, which however provides large difficulties in terms of precise and reproducible enzyme dosage. Additionally this is an extra process step which is economically not favourable.